The long-term objectives of this proposal are threefold: i) to determine the role of the CF gene product (CFTR) in modulating or mediating vesicle trafficking phenomena, such as exocytosis, in epithelial cells; ii) to test the hypothesis that plasma membrane Cl- permeability in epithelial cells is acutely regulated by a recycling of Cl- channels between cytoplasmic vesicles and plasma membrane and iii) to identify and, characterize additional Cl- channels within cytoplasmic vesicles whose properties and regulation may be distinct from those channels involved in controlling plasma membrane Cl- permeability. We will use a combination of optical, biochemical and biophysical techniques to address the following specific aims: 1) to develop and refine quantitative assays of endocytosis, exocytosis and plasma membrane recycling in secretory and reabsorptive epithelial cells; 2) to determine the general involvement of CFTR in mediating such vesicle trafficking phenomena; 3) to directly test the hypothesis that a vesicle trafficking phenomenon underlies the short-term regulation of epithelial Cl- permeability by hormones and autocoids and 4) to isolate specific vesicle populations and characterize the nature and regulation of their constituent Cl- channels. our results should provide significant insights into the function of CFTR in normal epithelial cells and the nature of the defective biochemical and physiologic events that are related to the defective Cl- transport seen in cystic fibrosis.